Purine nucleoside phosphorylase (PNP) is one of the enzymes comprising the “purine salvage pathway” and is responsible for the catalysis of the reversible phosphorolytic cleavage of purine ribonucleosides and 2′-deoxyribonucleosides. PNP is the only enzyme that degrades 2′-deoxyguansodine in T-cells, and when not degraded, 2′-deoxyguanosine is converted instead to 2′-deoxyguanosine triphosphate (dGTP), which accumulates. High levels of dGTP cause allosteric inhibition of ribonucleotide diphosphate reductase, which prevents the normal DNA replication required for clonal expansion of T-cell populations (Thelander, L. et al. Annu. Rev. Biochem., 1979, 48, 133). Genetic deficiency of PNP in humans results in the selective depletion of T-cells (Markert, L. M. Immunodefic. Rev., 1991, 3, 45). The pivotal role of PNP in T-cell proliferation has been demonstrated in patients with inherited PNP deficiency, where T-cell levels may be 1-3% of normal. This observation helped establish the critical role of PNP in T-cells and provided a rationale for developing inhibitors of PNP (Morris, P. E. et al., Current Pharmaceutical Design, 2000, 6, 943). PNP deficiency causes severe loss of T-cell mediated immunity while leaving B-cell immune response and other tissues unaffected (Giblett et al., Lancet, 1975, 1, 1010).
Undesirable activation of T-cells is associated with a number of human disease states, including Crohn's disease, psoriasis, rheumatoid arthritis, T-cell lymphomas, T-cell leukemia, solid tumors, transplant tissue rejection and possibly other autoimmune diseases (Evans, G. B. et al. Tetrahedron, 2000, 56, 3053).
Improving the delivery of drugs and other agents to target cells and tissues has been the focus of considerable research for many years. Though many attempts have been made to develop effective methods for importing biologically active molecules into cells, both in vivo and in vitro, none has proved to be entirely satisfactory. Optimizing the association of the inhibitory drug with its intracellular target, while minimizing intercellular redistribution of the drug, e.g., to neighboring cells, is often difficult or inefficient.
Most agents currently administered to a patient parenterally are not targeted, resulting in systemic delivery of the agent to cells and tissues of the body where it is unnecessary, and often undesirable. This may result in adverse drug side effects, and often limits the dose of a drug (e.g., glucocorticoids and other anti-inflammatory drugs) that can be administered. By comparison, although oral administration of drugs is generally recognized as a convenient and economical method of administration, oral administration can result in either (a) uptake of the drug through the cellular and tissue barriers, e.g., blood/brain, epithelial, cell membrane, resulting in undesirable systemic distribution, or (b) temporary residence of the drug within the gastrointestinal tract. Accordingly, a major goal has been to develop methods for specifically targeting agents to cells and tissues. Benefits of such treatment includes avoiding the general physiological effects of inappropriate delivery of such agents to other cells and tissues, such as uninfected cells.
Thus, there is a need for therapeutic agents that inhibit PNP with improved pharmacological properties, e.g., drugs having improved PNP-inhibitory activity and pharmacokinetic properties, including improved oral bioavailability, greater potency and extended effective half-life in vivo. Such inhibitors would have therapeutic potential as anti-inflammatory agents, immunosuppressants, and as anti-cancer agents. Specifically, such compounds may be used in the treatment of Crohn's disease, psoriasis, rheumatoid arthritis, T-cell lymphomas, T-cell leukemia, solid tumors, transplant tissue rejection and other autoimmune diseases. New PNP inhibitors should have fewer side effects, less complicated dosing schedules, and be orally active. In particular, there is a need for a less onerous dosage regimen, such as one pill, once per day.
Assay methods capable of determining the presence, absence or amounts of PNP inhibition are of practical utility in the search for inhibitors as well as for diagnosing the presence of conditions associated with PNP activity.